It is clear that the most effective means of immunization against cholera is prior history of clinical disease. The major objectives of this research proposal are to isolate and characterize, both genetically and biochemically, non-toxinogenic mutants of Vibrio cholerae that may serve as prototypes for the development of a live oral vaccine. The elucidation of the location and organization of the cholera toxin structural genes, the determination of the nature of cholera tox regulatory loci, the determination of the location of biosynthesis, mechanism(s) of secretion, assembly and release of hol cholera toxin from the bacterial cell are of prime importance in the development of effective and safe oral vaccines. The genetic stability of mutant strains will be determined by colonization of germ free rats and monitoring of feces for revertants. The germ free rat model system will be used for the development of in vivo bacterial genetic analysis.